1. Field of the Invention
The present invention relates to a method of enhancing the delivery of a nucleic acid into a cell. In particular are provided methods comprising forming a complex of a nucleic acid, a cationic liposome and a virus and administering the complex to the cell to enhance delivery of the nucleic acid to the cell. Also provided are complexes comprising a nucleic acid, a cationic liposome and a virus.
2. Background Art
Transfer of genes into cells and expression of the transferred genes therein has been a subject and goal of much study, with limited success. Several different methods have been reported in the literature for gene transfer in vitro and/or in vivo, some using viruses and/or liposomes.
The use of liposomes, and in particular, cationic liposomes, for gene transfer has been described in vitro (e.g., Ciccarone, V., et al., Focus, 15(3):73-83 (1992)). However, the efficiency of transfer recited in the literature has not been as high as desirable for effective gene therapy and has only been performed in a limited number of cell types.
Retroviruses have been administered in vitro with cationic liposomes to infect cultured cells and thus deliver the DNA of the retroviruses to the cells (Innes et al., J. Virol., 64(2):957-961 (1990)). Such a method requires insertion of the nucleic acid to be expressed into the viral genome, and further, viral genes are co-introduced into the target cells. Sendai virus (hemagglutinating virus of Japan (HVJ)) has been utilized wherein anionic or neutral liposomes encase a plasmid of choice, the plasmid-containing liposomes are then complexed with inactivated HVJ, and the liposome-HVJ complex is incubated with vascular smooth muscle cells to cause expression of the plasmid in the cells (Morishita et al., Hypertension, 21:894-899 (1993)). Forming these neutral or anionic liposomes that encase the DNA requires additional methods such as reverse-phase evaporation of various lipids, DNA, and any additional desired components of the liposomes, which method uses organic solvents (Kaneda, et al., Science, 243:375-378 (1989)). Additionally, the efficiency of these gene transfer methods is not very high.
Adenoviruses have also been utilized for gene transfer methods. Many of these methods, however, require the synthesis of a variety of molecular conjugates and/or the efficiency of transfer has not been as high as desired for effective gene therapy. Gao et al., Human Gene Therapy, 4:17-24 (1993) provides a method of in vivo delivery of genes to respiratory epithelium. This method requires the synthesis of two molecular conjugates, an adenovirus-polylysine conjugate joined by an antibody or chemical bridge and a DNA-polylysine conjugate, which are then complexed together to form an adenovirus-polylysine-DNA complex via the polylysine chains. This reference also disclosed the formation of a human transferrin-adenovirus-polylysine-DNA complex requiring the formation of different molecular conjugates which are then complexed together. Thus, Gao et al. creates a product having DNA linked to adenovirus via conjugation with polylysine without the use of liposomes.
Cristiano et al., utilizes adenovirus for gene delivery by forming an asialoorosomucoid (ASOR)-polylysine conjugate, which is then complexed with DNA. The method involves adding the complex to culture cells, followed immediately by the addition of replication-deficient adenovirus. The ASOR directs the complex specifically to hepatocytes. It is not clear whether a complex including the adenovirus can form in this in vitro method; however, if the method was repeated in vivo, because of the separate steps, the likelihood of complex formation would greatly decrease.
Yoshimura et al., describes a method wherein DNA and the cationic liposome LIPOFECTIN are complexed and then diluted. This diluted mixture is then added to cell cultures. Thirty minutes later, replication-deficient adenovirus is added to the cultures. Thus, the adenovirus is never complexed with the DNA. Furthermore, if the method were transferred to an in vivo use, also, no complex with the adenovirus could be formed because of the time lag. Such a method has been shown to provide some improvement in transfer efficiency in vitro.
It has been shown that empty adenoviral capsids bind double-stranded DNA (Gian, C. Z. and Tibbets, J. Virol. 32: 995-1005 (1979).
The present invention provides the use of replication-deficient adenovirus-DNA-cationic liposome complexes. The efficiency of gene transfer using the present inventive method is very high compared to other methods of gene transfer using liposome-DNA complexes or DNA-polylysine complexes. Given the simplicity of the complexing protocol for DNA-lipid-virus complexes (no genetic manipulations, no synthesis of molecular conjugates, no toxicity to cells), it has a wide applicability for transfecting genes transiently in vitro or in vivo. It is also much simpler to perform than earlier methods. The method thus fills a need for high-efficiency gene transfer techniques that many have previously tried to solve with only limited success.